Abstract: The abuse of antibiotics has caused severe environmental issues. Photocatalytic treatment technology has the advantages of high efficiency, strong effectiveness and no secondary pollution, which presents great potential for degrading or removing many kinds of environment pollutants. However, the intermediate products formed during the photocatalytic degradation process of antibiotics may possess greater toxicity than the antibiotics themselves, and the toxicity of most of these intermediates still remains unclear. Therefore, understanding the photocatalytic degradation pathways of antibiotics and obtaining qualitative and quantitative information about the main degradation products are of vital importance for the large-scale application of photocatalytic technology in the removal of antibiotics and other pollutants. Probe electrospray ionization mass spectrometry (PESI-MS) technology, adopting a solid probe as the electrospray ionization emitter, has the advantages of rapid response, low sample consumption, high salt tolerance, and strong resistance to suspended particles. These features make PESI-MS to be an ideal technique for direct analysis of complex systems, such as biological and chemical reactions. In this study, PESI-MS was used to perform continuous real-time in situ monitoring and analysis of the photocatalytic degradation of oxytetracycline (OTC), which photocatalyzed by TiO₂ under UV irradiation for 160 min, with spectra collected at a resolution of 1 spectrum per second. The results showed that the intensity of protonated OTC ions decreases progressively during the reaction, and forms product ions at various stages of degradation. 35 Kinds of key degradation products are identified by high time-resolved spectra. Based on the changes in the extracted ion chromatograms (EIC) of these products, 7 major degradation products are screened out and 10 possible degradation pathways are proposed, two of which can lead to the degradation of OTC into lower molecular weight products. Although complete photocatalytic degradation of OTC is not achieved in the experimental conditions, the amount of degradation products observed in this study is significantly higher than those previously reported using various high performance liquid chromatography mass spectrometry (HPLC-MS). Furthermore, in the early stages of the photocatalytic process, products of combined ethyl radicals and ethoxy radicals are detected, which likely are contributed by the ethanol added intentionally in the reaction. The use of organic solvents to reduce solution surface tension during PESI operation at atmospheric pressure is inevitable. In the future, we aim to develop a high-pressure PESI ionization source to resolve this issue, which is expected to reveal a more accurate photocatalytic reaction mechanism and pathway. The findings of this study provide valuable data for understanding the kinetics of photocatalytic reactions and developing new photocatalysts, helping researchers gain a more comprehensive understanding of photocatalytic mechanisms.